An analysis modality for vascular structures combining tissue-clearing technology and topological data analysis

The blood and lymphatic vasculature networks are not yet fully understood even in mouse because of the inherent limitations of imaging systems and quantification methods. This study aims to evaluate the usefulness of the tissue-clearing technology for visualizing blood and lymphatic vessels in adult mouse. Clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) enables us to capture the high-resolution 3D images of organ- or area-specific vascular structures. To evaluate these 3D structural images, signals are first classified from the original captured images by machine learning at pixel base. Then, these classified target signals are subjected to topological data analysis and non-homogeneous Poisson process model to extract geometric features. Consequently, the structural difference of vasculatures is successfully evaluated in mouse disease models. In conclusion, this study demonstrates the utility of CUBIC for analysis of vascular structures and presents its feasibility as an analysis modality in combination with 3D images and mathematical frameworks.

a The 3D images of the whole mouse and various organs in the control mice (ctrl). Normal mice (female, 4 months) were subjected to CUBIC procedures. The 3D images of the whole body, brain, heart, intestine, kidney, and lung are shown.
b The 3D and 2D (XY) images of VE-cad-tdTomato mice (2-4 months). The images of the brain, liver, and lung are shown.
c Simultaneous visualization of -SMA + and VE-cad + blood vessels in the heart. VE-cad-tdTomato mice (10-11 months) were sacrificed, and samples were subjected to CUBIC procedures. Samples were stained with anti--SMA-FITC antibody. The 3D whole-heart images and 2D (XY) images are shown. The enlarged 2D images of white insets are shown in the right panels (Z = 10 m step, digital zoom; 2.0).
d Expression of blood vasculature markers determined by 2D immunohistochemistry (IHC). Before sacrificing, tomato-lectin conjugated with FITC was injected in VE-cad-tdTomato mice (2-8 months), to which Tx were injected. The brain and stomach were excised. Frozen sections were stained with anti-CD31 antibody and encapsulated with a mounting agent containing DAPI. Tomato-lection conjugated with Texas-Red was injected into normal mice (3-4 months) before sacrifice, and samples were frozen. Frozen sections were stained with anti-CD31 antibody. Representative images (brain and stomach) are shown. a The 3D images of the intestine and mesentery in Prox1-GFP mice. The intestine with mesentery from Prox1-GFP mice (2-3 months) was embedded into 2% gel before RI adjustment (Z = 10 m step, digital zoom: 2.0).
b The 3D whole-organ images of the lymphatic vessels with VEGFR3 staining. Samples were stained with anti-VEGFR3 antibody. The 3D image of the stomach and the 2D image of the intestine are shown (Z = 10 m step, digital zoom; stomach: 1.25, intestine: 2.0).
c The signal confirmation of lymphatic vessels. The 3D images of the organs stained with anti-VEGFR3 antibody are shown. The stomach of Prox1-GFP mouse (3-4 months) was subjected to CUBIC procedures and stained with anti-VEGFR3 antibody (Z = 10 m step, digital zoom: 1.6).   Supplementary Fig. 3 Pixel classification and threshold of their probability.
The 3D whole-lung images (essentially the same with Fig. 3b and originated from control#1 in Fig. 7) from the original and classified signals at each threshold of probability (90%, 70%, 50%, 30%) shown in Figure 4b. The 3D images of whole-lung (a), enlarged 3D images (b) and 2D images (c) are shown. Yellow signals indicate the lymphatic vessels from the original images and green signals indicate the signals classified as lymphatic vessels.    Supplementary Fig. 4 Analysis of brain blood vessels using NHPP.
The classified signals as blood vessels were subjected to the NHPP analysis. The strength (a) of -SMA + blood vessels or VE-cad + blood vessels are shown in each brain area (a). The directionalities (b x , b y , b z ) of -SMA + blood vessels or VE-cad + blood vessels are shown in each brain area (b) (14 brain areas: cerebellum (CB), cortical subplate (CTXsp), fiber tracts (   The classified signals with different thresholds of probability (90, 70, 50, 30, 10%) shown in Supplementary Fig. 3 were analyzed with PH.
Persistent diagrams (PDs) are shown. The sample is originated from control#1 in Fig. 7. The red points (dimention1) and blue triangles (dimention2) represent planar feature points (loop) and spatial feature points (void), respectively, observed by the persistent homology method.  Supplementary Fig. 10 Analysis of lymphatic vessels and mouse melanoma B16F10 lung metastasis.

Supplementary Figure 8
a The distances between B16F10 cells and lymphatic vessels (pixel-based) are shown in each sample.
b Persistent diagram of the mouse lung lymphatic vessels in B16F10 experimental metastasis model as shown in Figure 7. The red points (dimention1) and blue triangles (dimention2) represent planar feature points (loop) and spatial feature points (void), respectively, observed by the persistent homology method. PDs are shown in each sample.